Flow-through Biosensor Research Articles

Diffusion–reaction modelling of DNA hybridization kinetics on biochips

An analytical expression for the rate of DNA hybridization on the surface of DNA biochips is established for the case of a finite 1-D diffusion space. The expression allows to account for the diffusion-limited supply of unreacted probe DNA and was obtained by solving the continuous 1-D reaction–diffusion mass balance using a Finite Fourier Transform technique. The extrapolation of the presently considered 1-D case to the full 3-D case is outlined as well. By bringing the obtained result into concurrence with the results of a stochastic random walk study, the kinetic constant k of the continuous reaction–diffusion model could be expressed as a function of the basic physico-chemical parameters (persistence length and jump frequency of the Brownian motion, collision frequency, binding probability) of the individual molecules. With the availability of an analytical expression describing the full time course of the 1-D hybridization process, and by using the Damköhler number Da= kh/ D mol, the different reaction- and diffusion-limited regimes occurring during the course of a hybridization process can now be described in general, dimensionless terms, allowing to establish some very simple rules for the design of DNA biochips and flow-through biosensors.

A lightweight measuring device for the continuous in vivo monitoring of glucose by means of ultraslow microdialysis in combination with a miniaturised flow-through biosensor

Background: Tight regulation of blood glucose levels from patients suffering from diabetes mellitus can significantly reduce the complications associated with this disease. For this reason, elaborate research efforts have been devoted to the development of a glucose sensor for the continuous in vivo monitoring of glucose. Although the use of microdialysis as a sampling interface between the body and the biosensor is widely accepted, a major drawback of conventional microdialysis is the limited in vivo recovery. Here, ultraslow microdialysis is proposed in order to obtain (near) quantitative in vivo recoveries. To avoid, however, unacceptable long delay times, the need for a small and low dead volume measuring device was recognised. Methods: A portable lightweight measuring device for continuous in vivo monitoring of glucose in subcutaneous tissue is presented. The measuring device consists of a miniaturised flow-through biosensor, connected to a microdialysis probe and a semi-vacuum pump. The biosensor is based on the amperometric detection of hydrogen peroxide after conversion of glucose by immobilised glucose oxidase. A portable potentiostat equipped with data logging is used for detection and registration. Results: The device was validated for its accuracy, precision, linearity, sensitivity, selectivity and stability during ex vivo and in vivo experiments. The linearity was found to be up to 30 mmol/l with a limit of detection of 0.05 mmol/l. The precision, depending on the biosensor tested was found to be 2–4%. No contribution to the signal could be observed from several tested electroactive species. The accuracy was found to be well in accordance with the criteria set for methods of Self Monitoring of Blood Glucose for patients with diabetes mellitus. The biosensors could be used for up to 3 days in the continuous mode. In vivo monitoring of glucose in dialysate of subcutaneous sampled tissue during glucose tolerance tests in healthy volunteers demonstrated the potential of this measuring device. Conclusions: A portable lightweight measuring device is presented which can measure continuously glucose in vivo without excessive calibration steps. The performance characteristics determined justify the application of this measuring device for the in vivo monitoring of glucose in subcutaneous sampled interstitium of diabetic patients.

Amperometric detection of glucose and ethanol in beverages using flow cell and immobilised on screen-printed carbon electrode PQQ-dependent glucose or alcohol dehydrogenases

Enzyme electrodes containing pyrroloquinoline quinone (PQQ)-dependent alcohol (ADH) or glucose dehydrogenase (GDH) as a biological component in combination with 4-ferrocenylphenol (FP) as electron transfer mediator between PQQ and a carbon surface were constructed. The transducer was based on screen-printed carbon electrode modified with FP and enzyme. The biosensors were installed in a flow cell for glucose and ethanol measurements. The calibration graphs were linear up to 10 mM glucose and 1 mM ethanol. To eliminate the electrode background current, 2,4,7-trinitro-9-fluorenone (TNF) was electrochemically polymerised on the carbon electrode surface. Due to this procedure, interfering compounds do not influence the electrochemical response of the sensor. The flow-through biosensor system was applied to measure the glucose and ethanol concentration in various beverages containing 0.3–200 mM glucose and about 2 M ethanol. The glucose biosensor data correlated with those obtained by refractometric measurements.

On-line continuous monitoring of glucose or lactate by ultraslow microdialysis combined with a flow-through nanoliter biosensor based on poly(m-phenylenediamine) ultra-thin polymer membrane as enzyme electrode.

A miniaturised flow-through biosensor with a cell volume of only a few nanoliters was developed in our laboratory. The biosensor can be directly coupled to a microdialysis or ultrafiltration probe. Sampling and continuous on-line monitoring can thus be carried out at submicroliter levels and as a consequence quantitative recoveries of the analyte of interest are achieved. Via this method excessive calibration procedures, as are necessary with conventional microdialysis, are avoided. Here, the construction and the performance of such a biosensor for the continuous on-line monitoring of glucose and lactate will be presented. The biosensor is based on the amperometric detection of hydrogen peroxide after conversion of the analyte of interest by an immobilised oxidoreductase enzyme. Immobilisation of the enzyme is performed through electropolymerisation of m-phenylenediamine. Strategies to improve the performance (e.g. linearity, selectivity and stability) of the miniaturised biosensor are discussed and ex vivo and in vivo experiments carried out thus far demonstrate the potential of this miniaturised flow-through biosensor.

Optical biosensors based on Prussian Blue films.

Optical biosensing schemes based on enzymatically modified inorganic/organic transparent films predominately composed of Prussian Blue are demonstrated. The composite film, which is non-electrochemically deposited on a non-conducting support. is used as an optical transducer for flow-through biosensors based on hydrolases and oxidases. Urease and glucose oxidase are utilized as model enzymes. Action of the urea biosensor is based on optical pH sensitivity of Prussian Blue indicator. The glucose biosensor is acting as first-generation optical biosensor based on in situ generated Prussian White transducer for hydrogen peroxide. These simple, single-pass transmission optical biosensors exhibit sensitivity in the millimolar range of concentration. The biosensors are very stable owing to presence of a poly(pyrrolylbenzoic acid) network in the composite material. This organic polymer plays a dual role as a binding agent for inorganic material and as a functionalized support for strong covalent immobilization of enzyme molecules.

Biosensors in Wine Production Monitoring

ABSTRACT An overview of probe-type and flow-through biosensors, together with biosensing systems, both batch and continuous biosensing systems, for monitoring typical species in wine processes is presented with the aim of showing the advantages and disadvantages involved in the use of each type of devices. Thus, biosensors and biosensing systems for the determination of ethanol (individual or together with that of other compounds), organic acids, glycerol, reducing sugars, acetaldehyde and sulfur dioxide/sulfite anions are reviewed and critically compared. The versatility and capability of continuous biosensing systems as compared with biosensors, particularly with probe-type biosensors working in a discontinuous, batch manner, is demonstrated.

Amperometric flow-through biosensor for the determination of cholinesterase inhibitors

An amperometric flow-through biosensor based on epoxy-carbon electrode and butyrylcholinesterase immobilised on nylon, cellulose nitrate or white tracing paper has been developed and examined for the determination of reversible and irreversible inhibitors. The analytical characteristics of inhibitor determination depend on the hydrophobicity of the membrane material. Flow-through biosensor with various enzymatic membranes makes it possible to determine fluoride in the concentration range 1×10 −4–25×10 −4 mol l −1 and 3.5×10 −5–1×10 −2 mol l −1 when cholinesterase solution is used. The analytical characteristics of fluoride determination do not differ significantly from those obtained in batch conditions. For diazinon the immobilisation of cholinesterase results in the decrease of detection limits from 5×10 −9 mol l −1 (native enzyme) to 4×10 −9 mol l −1 (nylon membrane) and 1.5×10 −9 mol l −1 (cellulose nitrate membrane). The influence of membrane material on analytical characteristics of FIA determination of inhibitors is due to the non-stationary distribution of reagents (fluoride) or sorptional preconcentration of the inhibitor (diazinon) in membrane.

Room temperature phosphorescence flow-through biosensing of anionic surfactants

The first flow-through biosensor for anionic surfactant determination based on a sensitive room temperature phosphorescence phase Al-Ferron coated with bovine serum albumin has been prepared, characterised and applied to the determination of sodium dodecylbenzenesulphonate in standard aqueous solution and in spiked water samples (lake, river and spring). The optosensor response to sodium dodecylbenzenesulphonate was linear from 5×10 −6 M to 5×10 −4 M which is a suitable range for the detection of anionic surfactant concentrations in polluted natural waters. The sample analysis was rapid (90% of the total response is reached in less than 30 s), convenient (no-sample pretreatment) and did not require organic reagents which are harmful to the environment. This work demonstrates the analytical potential of surfactant–protein interactions as a biorecognition mechanism using suitable luminescence probes.

General Fluorimetric Flow-through Sensor for the Determination of Oxidase Substrates

A new flow-through sensor for the determination of hydrogen peroxide based on the dual immobilisation of the catalyst and the reaction product (permanent immobilisation in a reactor and transient retention in the flow-cell, respectively) is proposed. The support packed in the flow-cell is an aminopropyl-bonded silica gel non-fluorescent material. The approach provides a linear range between 0.03–17 ng ml–1 H2O2 (r2 = 0.9954, RSD values less than 3.5%). This sensor is 80 times more sensitive than a photometric flow-through biosensor previously reported and based on similar principles.

Use of mathematical models to describe dynamic behaviour of potentiometric biosensors: Comparison of deterministic and empirical approaches to an urea flow-through biosensor

The response of a flow-through biosensor is the consequence of complex interrelations between the dilution of the sample in a continuous stream (governed by physical variables) and the phenomena involved in the reactions between enzyme and substrate (governed by diffusion and kinetic processes). These interrelations make it difficult to know in advance the influence of manifold parameters in the biosensor response. If a reliable model of these processes is attainable, it will be possible to test rapidly, by computer, the effect of the variation of different operating variables on the response of the biosensor. This paper presents the results obtained when two models are applied to describe dynamic behaviour of a potentiometric flow-through urea biosensor. One of these models uses an empirical approach based on neural networks, while the other proposes a deterministic approach that takes into account reaction-diffusion equations.

Spain - Development of an optical flow-through biosensor for sulphite in environmental samples: In ANAL. CHIM. ACTA (311/3 (281–287) 1995) M.D. Luque De Castro & J.M. Fernandez-Romero of the University of Cordoba report on ‘Development of an optical flow-through biosensor for the determination of sulphite in environmental samples’

Spain - Development of an optical flow-through biosensor for sulphite in environmental samples: In ANAL. CHIM. ACTA (311/3 (281–287) 1995) M.D. Luque De Castro & J.M. Fernandez-Romero of the University of Cordoba report on ‘Development of an optical flow-through biosensor for the determination of sulphite in environmental samples’

Spain - Pesticide determination biosensor: In ANAL. CHIM. ACTA (308/1–3 (129–136) 1995) C. La Rosa, F. Pariente, L. Hernandez & E. Lorenzo of Universidad Autonoma, Madrid report on ‘Amperometric flow-through biosensor for the determination of pesticides’

Spain - Pesticide determination biosensor: In ANAL. CHIM. ACTA (308/1–3 (129–136) 1995) C. La Rosa, F. Pariente, L. Hernandez & E. Lorenzo of Universidad Autonoma, Madrid report on ‘Amperometric flow-through biosensor for the determination of pesticides’

Development of an optical flow-through biosensor for the determination of sulphite in environmental samples

A continuous-flow biosensor arrangement based on the dual immobilization of a biocatalyst on controlled-pore glass and the reaction product on a resin support both packed in the flow-cell of a photometric detector for the determination of sulphite is proposed. The method thus developed affords low detection and quantitation limits (3 and 10 ng ml −1 of sulphite, calculated as ±3 σ and ±10 σ, respectively, for n = 20), wide linear range (10–1000 ng ml −1 of sulphite), good precision (R.S.D. = 2.6% for 60 ng ml −1 and 0.7% for 200 ng ml −1 of the analyte), and a sampling frequency of 16 h −1. The hydrodynamic assembly has been applied to the determination and recovery of the analyte in spiked, aqueous samples with excellent results.

Amperometric flow-through biosensor for the determination of pesticides

An amperometric flow-through biosensor for the determination of pesticides is proposed. It is based on the inhibition of the acetyl cholinesterase-catalysed hydrolysis of 4-aminophenylacetate. The calibration graphs were linear from 5.0 × 10 −7 to 1.0 × 10 −5 M and 5.0 × 10 −7 to 5.0 × 10 −5 M for paroxon and carbaryl, respectively. The detection limit (5% of inhibition) was 1.0 × 10 −7 M pesticide. The relative standard deviations (R.S.D.) ( n = 5) were 3.7% for 4.0 × 10 −5 M and 4.0% for 8.0 × 10 −6 M for either carbaryl or paroxon. Electroactive species such as uric and ascorbic acid and benzaldehyde that could be oxidized at the same potential as 4-aminophenol, do not interfere. However, compounds which strongly absorb onto the electrode surface, such as bovine serum albumin (BSA) and surfactants capable of denaturing the enzyme activity, cause an interference. The stability of the sensor was high and even after repetitive use for one month the electrode retained 90% of its original activity. The determination of carbaryl and paroxon was carried out in lagoon water and kiwi fruits. The lowest concentration of pesticide determined in lagoon water was 1.0 × 10 −7 M for paroxon and 4.0 × 10 −7 M for carbaryl.

Flow-through biosensor for sequential determination of total and prostatic acid phosphatase activity

Two continuous methods based on the use of conventional flow-injection system and a flow-through sensor for the sequential determination of total and prostatic acid phosphatase activity are reported. The biochemical basis of both methods is the hydrolysis of p-nitrophenyl-phosphate catalysed by the analyte and photometric monitoring of the reaction product ( p-nitrophenol) at 405 nm. The enhancement of sensitivity achieved when this species is retained on the support packed in the flow cell makes the method suitable for the determination of the analyte activity in serum samples after 1:5 dilution. The excellent agreement of the results obtained by the proposed and a conventional method and also the recoveries obtained prove the usefulness of the proposed biosensor.

Fluorimetric determination of alkaline phosphatase activity in human serum by use of a flow-through biosensor

A fluorimetric method for the determination of alkaline phosphatase activity based on the use of a flow-through biosensor is reported. The biochemical basis of the method is the hydrolysis of 4-methyl-umbelliferone-phosphate catalyzed by the analyte with fluorimetric monitoring of the 4-methyl-umbelliferone formed ( λ ex = 365 nm, λ em = 445 nm). The enhancement of sensitivity achieved when the reaction product is retained on the support packed in the flow-cell makes the method suitable for determination of the analytes in serum samples after 1:50 dilution. The linear range is found to be between 0.1 and 20 U1 −1, with relative standard deviation less than 2.2%. The use of this biosensor was tested by the determination of the analyte in human serum from healthy and sick individuals with excellent recoveries (94–103%).

Determination of Enzymatic Activities Based on an Optical Flow-Through p-Nitrophenol Sensor

Abstract An optical flow-through biosensor based on the transient retention of p-nitrophenol on an ion-exchange support packed in the flow cell is presented. The approach was applied to the determination of the activity of some enzymes which catalyze the conversion of p-nitrophenyl-derivatives into p-nitrophenol. The method was first developed for determination of p-nitrophenol yielding a linear range between 0.1–5 μg/mL (r2 = 0.9922, r.s.d.% less than 2.5), then, it was also applied to the determination of β-D-glucuronidase activity in serum, with a linear range between 0.1–20 U/L (r2 = 0.9976, r.s.d.% less than 3.0), and a sampling frequency of 20 h−1. The application of the method to the determination of the enzyme activity in serum samples provided results consistent with those obtained by a conventional method and recoveries within 95–104%.

Reaction-rate measurements by use of membraneless flow-through biosensors

Three different approaches to performing reaction-rate measurements by using flow-through optical biosensors are proposed. This type of biosensor consists of a special flow-cell located in a conventional spectrophotometer, on the inner walls of which a biocatalyst is immobilized. Reaction-rate measurements can be made by coupling the biosensor on-line to different flow-injection (FI) manifolds: a normal FI system in which the flow is halted while the sample plug is in the flow-cell; another where the flow direction is changed iteratively; and an open-closed configuration. The performance of the three manifolds is critically studied and compared by using the same biosensor (immobilized β- d-glucuronidase) for the determination of glucuronide derivatives.